beta-defensin-2 and beta-defensin-3 reduce intestinal

Research ArticleBeta-Defensin-2 and Beta-Defensin-3 Reduce IntestinalDamage Caused by Salmonella typhimurium Modulating theExpression of Cytokines and Enhancing the Probiotic Activity ofEnterococcus faecium

Alessandra Fusco, Vittoria Savio, Marcella Cammarota, Alberto Alfano,Chiara Schiraldi, and Giovanna Donnarumma

Department of Experimental Medicine, University of Campania “Luigi Vanvitelli”, Via De Crecchio No. 7, 80138 Naples, Italy

Correspondence should be addressed to Alessandra Fusco; [email protected] andGiovanna Donnarumma; [email protected]

Received 29 May 2017; Revised 9 August 2017; Accepted 5 September 2017; Published 9 November 2017

Academic Editor: Mitesh Dwivedi

Copyright © 2017 Alessandra Fusco et al. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the originalwork is properly cited.

The intestinal microbiota is a major factor in human health and disease. This microbial community includes autochthonous(permanent inhabitants) and allochthonous (transient inhabitants) microorganisms that contribute to maintaining the integrityof the intestinal wall, modulating responses to pathogenic noxae and representing a key factor in the maturation of the immunesystem. If this healthy microbiota is disrupted by antibiotics, chemotherapy, or a change in diet, intestinal colonization bypathogenic bacteria or viruses may occur, leading to disease. To manage substantial microbial exposure, epithelial surfacesof the intestinal tract produce a diverse arsenal of antimicrobial peptides (AMPs), including, of considerable importance,the β-defensins, which directly kill or inhibit the growth of microorganisms. Based on the literature data, the purpose of thiswork was to create a line of intestinal epithelial cells able to stably express gene encoding human β-defensin-2 (hBD-2) andhuman β-defensin-3 (hBD-3), in order to test their role in S. typhimurium infections and their interaction with the bacteria ofthe gut microbiota.

1. Introduction

The gastrointestinal tract is the most important immuneorgan of the human body. The intestinal surface has a strate-gic position at the interface between the antigenic luminalenvironment and the internal milieu of the host and isconstantly exposed to various antigens from food or fromdifferent pathogens.

The human intestine hosts a large and diverse microbialcommunity and contains approximatively 400–1000 differ-ent species of bacteria, virus, and fungi. These microbes arecollectively referred to as the commensal microbiota.

The importance of the homeostatic maintenance ofhuman health by the intestinal microbiota has become atopic of great interest [1–4]. Commensal bacteria modulate

the expression of genes involved in several major intestinaland extraintestinal functions, including the xenobioticmetabolism, postnatal intestinal maturation, nutrientabsorption, and fortification of the mucosal barrier, andinhibit the growth of pathogenic species through the produc-tion of antimicrobial substances. In addition, the humanmicrobiota is involved in the synthesis of essential aminoacids and vitamins (K, B2, B1, B6, B12, folic acid, biotin,and pantothenic acid) in the absorption of calcium, magne-sium, and iron, in the extraction of energy from componentsin the diet, and in the regulation of fat storage [5, 6].

The genus Enterococcus is a group of lactic acid bacteria(LAB) whose use as probiotic microorganisms is controver-sial [7] as they are sometimes associated with infections inhumans [8–11]. However, it has been shown that several

HindawiJournal of Immunology ResearchVolume 2017, Article ID 6976935, 9 pageshttps://doi.org/10.1155/2017/6976935

https://doi.org/10.1155/2017/6976935

enterococcal strains, which may rebalance the intestinal bac-terial flora in antibiotic-induced dysbiosis [12], can intervenein the antitumoral protective response [13] and can haveantiviral activity [14].

Of great interest is Enterococcus faecium, for which theEuropean Food Safety Authority (EFSA) has recently estab-lished new guidelines for distinguishing between beneficialor potentially pathogenic strains based on their susceptibilityto ampicillin and on the presence of specific genetic markersof virulence (esp, hylEfm, IS16).

It has been demonstrated that the culture supernatant ofthe E. faecium strain in the human intestinal epithelial cellshas a strong bactericidal effect on enteroaggregative Escheri-chia coli, including the induction of membrane damage andcell lysis [15]. The ability of these bacteria to produce enter-ocins is remarkable, and these instead can be applied as foodbiopreservatives [16, 17]. In fact, E. faecium RZS C5, a natu-ral cheese isolate, has a strong activity against Listeria mono-cytogene adhesion and invasion of Caco-2 cells [18].

E. faecium SF68® (NCIMB 10415) is present in pharma-ceutical preparations as a feed additive for different animals[19, 20], since it is capable of lowering the bacterial concen-tration of E. coli and stimulates an anti-inflammatoryresponse [21].

Therefore, the human intestinal microbiota contributesto maintaining the integrity and impermeability of the intes-tinal wall, which represents the first line of defense againstpathogens. Among these, Salmonella enterica serovar typhi-murium (S. typhimurium) is one of the most common nonty-phoidal Salmonella (NTS) considered a major cause of acutefood infection [22]. This Gram-negative bacillus can causesevere diarrhoea, vomiting, fever, and death in severe cases,especially in children, the elderly, and immunocompromisedpatients.

S. typhimurium can survive and replicate within hostmacrophages and induces the activation of NF-kB and thesecretion of proinflammatory cytokines, such as interleukin-(IL-) 8 [23] and tumor necrosis factor alpha (TNF-α) [24].This inflammation also helps it to compete with the microor-ganisms of the host microbiota [25].

Probiotics attenuate NF-kB activation and inflammatorycytokine production in the intestinal epithelial cells in vitro[26, 27] and in vivo [28–30].

In addition to serving as a protective barrier, the intes-tinal epithelium plays an active role in the intestinalimmune response through the secretion of inflammatorycytokines, chemokines, and antimicrobial peptides suchas β-defensins [31].

The family of β-defensins is composed of small cationicpeptides produced by epithelial cells, Paneth cells, neutro-phils, and macrophages, constitutive or induced by microor-ganisms or cytokines that contribute to the broad spectruminnate immunity.

Human β-defensin-2 (hBD-2) is an inducible antimicro-bial peptide with a molecular mass of 4–6 kD and acts as anendogenous antibiotic in the defense against Gram-negativebacteria, among which the potential pathogenic microbes ofthe gut [32, 33], and can be induced by endogenous stimuli,infections, or wounds.

Human β-defensin-3 (hBD-3) is identified in psoriaticscales [34] and is expressed in the skin, placenta, and oraltissue [34, 35] and shows antimicrobial activity againstGram-positive and Gram-negative bacteria and fungi. Beinginsensitive to high salt concentrations, its antimicrobialactivity results to be greater than that of hBD-2 [36].

Both hBD-2 and hBD-3 are chemoattractants for neutro-phils [37] and memory T-cells, induce histamine releasefrom mast cells and prostaglandin synthesis, and play a rolealso in allergic responses.

In the light of the growing interest of the use of antimi-crobial peptides as natural defense molecules against patho-gens and due to the increased antibiotic resistance by anumber of pathogenic bacteria, this study aims to create aline of intestinal epithelial cells expressing high concentra-tions of the antimicrobial peptides hBD-2 and hBD-3 andto assess their role in the host inflammatory response result-ing from bacterial infections.

2. Materials and Methods

2.1. Cloning. Total RNA was extracted using a High PureRNA Isolation Kit (Roche Diagnostics) from primary cul-tures of human keratinocytes stimulated with the LPS ofPseudomonas aeruginosa and TNF-α in order to obtain ahigh production of antimicrobial peptides. It was subse-quently transcribed into complementary cDNA using ran-dom hexamer primers (Random hexamers, Roche) at 42°Cfor 45 minutes, according to the manufacturer’s instructions.Two pairs of degenerate primers, designed on their specificamino acid sequence (hBD-2 for 5′-CCAGCCATCAGC-CATGAGGGT-3′, hBD-2 rev-5′-GGAGCCCTTTCTGAATCCGCA-3′ 254 bp; and hBD-3 for 5′-CGGCAGCATTTTGCGCCA-3′, hBD-3 rev 5′-CTAGCAGCTAT-GAGGATC-3′), were used to amplify, by RT-PCR, gene cod-ing hBD-2 and hBD-3 with FastStart High Fidelity (RocheDiagnostics). The amplification programs were the following:35 cycles at 94°C for 1′, 63°C (for hBD-2) or 58°C (for hBD-3)for 1′, and 72°C for 1′; the PCR products were 254 and 206base pairs.

The amplified DNA fragments were subjected to restric-tion and sequencing analysis and cloned into the pEF/V5-HIS TOPO (Invitrogen) vector using the T4 DNA ligase(Invitrogen), in accordance with the manufacturer’s proto-col, and then transformed into E. coli TOP 10 (Invitrogen).

The cloning vectors, pEF/V5-HIS TOPO-hBD-2 andpEF/V5-HIS TOPO-hBD-3, were extracted from the bacte-rial culture and amplified using a QIAprep Spin MidiprepKit (QIAGEN).

2.2. Transfection. Caco-2 cells were transfected using theIBAfect reagent (IBA), according to the manufacturer’s man-uals. Briefly, 3× 105 cells were seeded in 6-well plates, andimmediately after seeding, plasmids conjugated with thetransfection reagent were added. The mixture was incubatedfor 24 and 48 hours. After incubation, the success of theexperiment was verified by the extraction of mRNA from

2 Journal of Immunology Research

treated cells and by the amplification of hBD-2 and hBD-3genes by PCR.

Cell-free supernatants of the transfected cells were recov-ered by centrifugation and assayed for the hBD-2 and hBD-3concentration by an enzyme-linked immunosorbent assay(Phoenix Pharmaceuticals Inc.).

For blasticidin selection, untransfected and transfectedcells were cultured at 37°C and 5% CO2 for 14 days in thepresence of the following increasing concentrations of blasti-cidin S (Sigma-Aldrich): 5, 10, 20, 50, 100, and 200 μg/ml.Then, MTT-labelling reagent was added at a final concentra-tion of 0.5mg/ml. After 4 hours, a solubilization solution wasadded to each well and the plates were incubated overnight.Spectrophotometric absorbance was measured using amicroplate (ELISA) reader at a wavelength of 570nm.

2.3. Bacterial Strains. S. enterica subsp. enterica serovar typhi-murium (ATCC® 14028GFP™) was cultured on Luria-Bertani agar (Oxoid, Unipath, Basingstoke, UK). E. faecium(ATCC 27270™) was cultured on Bacto Tryptic Soy agar(TSA, Difco Laboratories). These strains were grown at37°C for 18h.

2.4. Cell Culture and Infection. Caco-2 cells (human Cauca-sian colon adenocarcinoma cells) were routinely cultured inDulbecco’s modified eagle medium (DMEM, Gibco) supple-mented with 1% Penstrep, 1% glutamine, and 10% fetal calfserum (Invitrogen) at 37°C at 5% CO2. After transfection,the cells were grown in a sterile 75 cm2

flask at a concentra-tion of 3× 105 to confluence for 21 days to reach full differen-tiation and polarization. The culture medium was changedevery two days.

Subsequently, fully differentiated cells were seeded intosix-well plates and then infected with exponentially growingbacteria at a multiplicity of infection (MOI) of 100 for 6hours (for gene expression analysis) and 24 h (for ELISAassay) at 37°C in 5% CO2 in DMEM without antibiotics. Inthe case of coinfection, preincubation of one hour with E. fae-cium was followed by the addition of S. typhimurium withoutthe removal of the probiotic bacterium.

At the end of the experiment, bacteria present in thesupernatants of infected and coinfected cells were counted(CFUs) by spreading serial dilutions on selective mediumHiCrome™ E. faecium Agar Base (Sigma-Aldrich) andBrilliance Salmonella agar (OXOID) and were incubatedat 37°C overnight.

2.5. Real-Time PCR. In order to evaluate the expression ofpro- and anti-inflammatory cytokines, the cells at the endof treatments were washed three times with sterile PBS, andthe total RNA was extracted using High Pure RNA IsolationKit (Roche Diagnostics).

Two hundred nanograms of total cellular RNA werereverse transcribed (Expand Reverse Transcriptase, Roche)into complementary DNA (cDNA) using random hexamerprimers (Random hexamers, Roche) at 42°C for 45 minutes,according to the manufacturer’s instructions [38]. Real-timePCR for IL-6, IL-8, TNF-α, IL-1α, IL-1β, and TGF-β was car-ried out with the LC FastStart DNA Master SYBR Green kitusing 2 μl of cDNA, corresponding to 10 ng of total RNAin a 20ml final volume, 3mM MgCl2, and 0.5mM senseand antisense primers (Table 1). After amplification, meltingcurve analysis was performed by heating to 95°C for 15 s witha temperature transition rate of 20°C/s, cooling to 60°C for15 s with a temperature transition rate of 20°C/s, and thenheating the sample at 0.1°C/s to 95°C. The results were thenanalyzed using LightCycler software (Roche Diagnostics).The standard curve of each primer pair was establishedwith serial dilutions of cDNA. All PCR reactions wererun in triplicate. The specificity of the amplification prod-ucts was verified by electrophoresis on a 2% agarose geland visualization by ethidium bromide staining.

2.6. ELISA Assay for Pro- and Anti-Inflammatory Cytokines.Caco-2 cell monolayers were infected with S. typhimuriumand/or E. faecium for 24h at 37°C, as described above. Atthe end of the experiment, supernatants were harvested andthe presence of cytokines IL-6, IL-8, IL-1β, TNF-α, andTGF-β was analyzed by enzyme-linked immunosorbentassay (ELISA, ThermoFischer Scientific Inc.).

Table 1: Primer sequences and amplification programs.

Gene Primer sequence Conditions Product size (bp)

IL-65′-ATGAACTCCTTCTCCACAAGCGC-3′5′-GAAGAGCCCTCAGGCTGGACTG-3′ 5″at 95°C, 13″ at 56°C, and 25″at 72°C for 40 cycles 628

IL-85′-ATGACTTCCAAGCTGGCCGTG-3′

5′-TGAATTCTCAGCCCTCTTCAAAAACTTCTC-3′ 5″at 94°C, 6″ at 55°C, and 12″at 72°C for 40 cycles 297

IL-1β5′-GCATCCAGCTACGAATCTCC-3′5′-CCACATTCAGCACAGGACTC-3′ 5″at 95°C, 14″ at 58°C, and 28″at 72°C for 40 cycles 708

TGF-β5′-CCGACTACTACGCCAAGGAGGTCAC-3′5′-AGGCCGGTTCATGCCATGAATGGTG-3′ 5″at 94°C, 9″ at 60°C, and 18″at 72°C for 40 cycles 439

IL-1α5′-CATGTCAAATTTCACTGCTTCATCC-3′5′-GTCTCTGAATCAGAAATCCTTCTATC-3′ 5″at 95°C, 8″at 55°C, and 17″at 72°C for 45 cycles 421

TNF-α5′-CAGAGGGAAGAGTTCCCCAG-3′5′-CCTTGGTCTGGTAGGAGACG-3′ 5″at 95°C, 6″ at 57°C, and 13″at 72°C for 40 cycles 324

3Journal of Immunology Research

2.7. Bacterial Internalization Assay. Untransfected Caco-2cell cultures were infected with S. typhimurium alone or coin-fected with S. typhimurium and E. faecium as previouslydescribed. In another set of experiments, E. faecium was heatkilled by incubating at 60°C for 45min and subcultured onTSA plates (Difco Laboratories) overnight at 37°C to provethat no viable organisms remained. Killed bacterial prepara-tion was resuspended in DMEM without antibiotics andadded to cell monolayer an hour before the addition of S.typhimurium. After 2 h of incubation at 37°C, infected mono-layers were extensively washed with sterile PBS and furtherincubated for another two hours in the DMEM medium,and supplemented with gentamicin sulphate (250 μg ml-1)(Sigma-Aldrich) in order to kill the extracellular bacteria.At the end of the experiments, infected monolayers wereextensively washed in PBS then lysed with a solution of0.1% Triton X-100 (Sigma-Aldrich) in PBS for 10 minutesat room temperature to count internalized bacteria. Aliquotsof cell lysates were serially diluted and plated on BrillianceSalmonella agar (OXOID) and incubated at 37°C overnightto quantify viable intracellular bacteria (CFUs/ml). Theefficiency was calculated as the ratio of the number of cell-

internalized bacteria with the number of bacteria used toinfect the cell monolayers.

2.8. Statistical Analysis. Significant differences among groupswere assessed through two-way ANOVA by using GraphPadPrism 6.0. The data are expressed as means± standard devia-tion (SD) of three independent experiments.

3. Results

3.1. Cloning and Transfection. The hBD-2 and hBD-3 geneswere successfully amplified by RT-PCR from a total cellularRNA. As expected, the PCR products were 254 and 206 bpin length. These products were inserted with high efficiencyin the pEF/V5-HIS TOPO vector.

The success of transfection of the cloning products incolorectal adenocarcinoma Caco-2 cells was verified after24 and 48 hours by RT-PCR and after 48 hours by ELISAassay on cell supernatants (Figure 1).

3.2. Blasticidin Selection and Cellular Viability. The toxicitycurve performed on transfected and untransfected cells

1 2 3 M 4 5 6

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Figure 1: (a) hBD-2 mRNA expression in untransfected cells (lane 1), 24 hours (lane 2), and 48 hours (lane 3) after transfection; hBD-3mRNA expression in untransfected cells (lane 4), 24 hours (lane 5), and 48 hours (lane 6) after transfection. (b) hBD-2 concentration incell supernatants 48 hours after transfection. (c) hBD-3 concentration in cell supernatants 48 hours after transfection.


showed that the optimal antibiotic concentration for theselection of stable clones was 200 μg/ml. These data werealso supported by the results of the cellular viability assay(see Supplementary Material available online at https://doi.org/10.1155/2017/6976935). The selected clones were thencultured for an additional 21 days to obtain their differen-tiation, which was characterized by polarization and theformation of microvilli.

3.3. Evaluation of the Host Inflammatory Response. After theinfection of untransfected and hBD-2- or hBD-3-transfectedCaco-2 cells with E. faecium and/or S. typhimurium, weexamined the host response by evaluating the expression ofproinflammatory cytokines IL-6, IL-8 Il-1α, and IL-1β andanti-inflammatory cytokine TGF-β by real-time PCR.

Thedataobtainedshowedthatthecells transfectedwiththehBD-2 and hBD-3 genes and infected with S. typhimuriumshowed a lesser expression of proinflammatory cytokinescompared to the untransfected control. Instead, an infec-tion of Caco-2 cells with E. faecium resulted only in aslight increase of expression of proinflammatory cytokinesand an increase in anti-inflammatory cytokine TGF-β,which was more apparent in the presence of antimicrobialpeptides; these data confirm that E. faecium did not act asa pathogen and did not induce an increase in the inflam-matory response (Figure 2).

In addition, during the coinfection with S. typhimuriumand E. faecium, the already significant decrease in expressionlevels of proinflammatory cytokines revealed in the trans-fected cells during infection with S. typhimurium alone is evenmore pronounced, indicating that the antimicrobial peptideshave enhanced probiotic antibacterial activity (Figure 3).

These data were also confirmed by ELISA protein assay.

3.4. Evaluation of Bacteria Viability. In order to test thetoxicity of antimicrobial peptides against S. typhimuriumand E. faecium, the supernatants of the coinfected cells weresubjected to serial dilutions and plated on selective media.

Our results indicate that both hBD-2 and hBD-3 possessselective toxicity towards S. typhimurium and did not inter-fere with the growth of E. faecium (Table 2).

3.5. Effect of E. faecium and AMPs on S. typhimuriumInvasiveness. Preincubation of untransfected Caco-2 cellswith live E. faecium significantly affected S. typhimuriuminternalization, reducing it by 45.8%. Conversely, pretreat-ment with heat-killed E. faecium does not interfere with theinvasive capacity of the pathogen (Figure 4).

4. Discussion

Innate immunity, in particular through antimicrobial pep-tides (AMPs), plays a key role in maintaining the balancebetween protection against pathogens and normal microbialtolerance; AMPs are structurally heterogeneous peptides ofamphipathic nature isolated from a wide variety of organ-isms, plants, insects, amphibians, and mammals that are ableto kill bacteria, fungi, and viruses quickly. Among these, thehuman β-defensins have received considerable interest.These peptides are produced by epithelial cells, constitu-tively, or as a result of certain stimuli such as microorganismsor cytokines. Defensins are able to attract inflammatory cellssuch as neutrophils, T cells, macrophages, and epithelialcells capable of releasing inflammatory mediators such asIL-6, IL-8, and IL-1β, as well as destabilizing microbialmembranes; moreover, they have the ability to remodelthe tissues and bind LPS.

S. typhimurium versus E. faecium

1200

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Untransfected +S. typhimurium

IL-1�훽

IL-6

IL-8

TNF-�훼

TGF-�훽

11001000

900800700600500400300200200150100

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hBD-2 + S. typhimuriumhBD-3 + S. typhimurium

Untransfected +E. faeciumhBD-2 + E. faeciumhBD-3 + E. faecium

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S. typhimurium versus E. faecium1200

% m

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exp

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IL-1�훽

IL-6

IL-8

TNF-�훼

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11001000

900800700600500400300200200150100

500

Untransfected +S. typhimuriumHBD-2 + S. typhimuriumHBD-3 + S. typhimurium

Untransfected +E. faeciumHBD-2 + E. faeciumHBD-3 + E. faecium

(b)

Figure 2: Comparison between relative gene expression (a) and protein concentration (b) in Caco-2 cells infected with S. typhimurium andCaco-2 cells infected with E. faecium. Data are mean± SD and are expressed as the percentage of increment compared to uninfected controls.


https://doi.org/10.1155/2017/6976935

https://doi.org/10.1155/2017/6976935

In particular, β-defensin-2 (hBD-2) and β-defensin-3(hBD-3) are present in various epithelia, such as skin [39],oral cavity [40], paranasal sinuses, gingival [41], corneal[42], intestinal, respiratory, and urogenital epithelium [31],and show antimicrobial activity against Gram-positive andGram-negative bacteria and fungi.

It has been estimated that the number of microbespresent throughout the human body amounts to approxi-mately 100 trillion cells, tenfold the number of humancells, and suggested that they encode 100-fold more

unique genes than our own genome [43]. Most of themare components of the gut microbiota, which containsbetween 1000 and 1150 prevalent bacterial species thatplay a central role in human health [43, 44].

This community is defined as a “metabolic organ,” as itplays a primary role in maintaining homeostasis by interven-ing in the regulation of metabolism and nutritional, physio-logical, and immunological functions.

In the first phase of this work, we worked on creating,by cloning and gene transfection techniques, a line of

S. typhimurium versus S. typhimurium + E. faecium

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Untransfected + S. typhimurium +E. faeciumhBD-2 + S. typhimurium

hBD-2 + S. typhimurium +E. faeciumhBD-3 + S. typhimuriumhBD-3 + S. typhimurium +E. faecium

(a)

S. typhimurium versus S. typhimurium + E. faecium

% p

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IL-6

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Untransfected + S. typhimuriumUntransfected + S. typhimurium +E. faeciumhBD-2 + S. typhimurium

hBD-2 + S. typhimurium +E. faeciumhBD-3 + S. typhimuriumhBD-3 + S. typhimurium +E. faecium

(b)

Figure 3: Comparison between relative gene expression (a) and protein concentration (b) in Caco-2 cells infected with S. typhimurium aloneand Caco-2 cells coinfected with S. typhimurium and E. faecium. Data are mean± SD and are expressed as the percentage of incrementcompared to uninfected controls.


intestinal epithelial cells (Caco-2 cells) that expresses hBD-2 and hBD-3 genes. This allowed us to evaluate the role ofthese peptides in protecting the intestinal epitheliumagainst S. typhimurium, alone or in cooperation with E.faecium, which is one of the major components of thehuman gut microbiota [45, 46].

The first data obtained from the CFUs/ml counts follow-ing infection and coinfection showed that there was a markedreduction in the number of colonies of S. typhimurium com-pared to untransfected cells in transfected cells, while thenumber of colonies of E. faecium remained unchanged,which shows that the antimicrobial peptides selectively car-ried out their microbicidal activity against the pathogen.

Mucosal surfaces are lined with epithelial cells that form abarrier between potentially pathogenic microorganisms andthe host tissues. Penetration of this layer by invasive bacteriainitially leads to an acute inflammatory response, a hallmarkof which is the local accumulation of polymorphonuclear

leukocytes. After the infection with pathogenic bacteria,epithelial cells at mucosal surfaces can secrete chemicalmediators, such as proinflammatory and chemoattractantcytokines constituting surveillance and warning system forthe immune and inflammatory cells present in the underly-ing mucosa [47]. However, in sites where there is a physio-logically high bacterial concentration due to the residentmicrobial flora, that is, the colon, cytokine production isclosely dependent on bacterial invasiveness, as only invasivebacteria induce cytokine secretion [48–50].

Among these, IL-6, IL-1, and TNF-α are highly expressedin most inflammatory states so as to often be considered atarget of therapeutic intervention.

IL-8 chemokine is also thought to be an early signal ofacute inflammation, as it is secreted by the intestinal epithe-lial cells following bacterial invasion, and accumulates inthe mucosa underlying the epithelial cell layer where theIL-8 responsive effector cells reside. In addition, it has beenshown that the presence of the IL-8 in serum is a diagnosticmarker for neonatal bacterial infection [51, 52].

The results obtained show that the inflammatoryresponse in hBD-2- and hBD-3-transfected cells is modifiedwith respect to untransfected cells, since the expression ofproinflammatory cytokines IL-6, IL-8, TNF-α, IL-1α, andIL-1β is greatly reduced, while the expression of anti-inflammatory cytokine TGF-β is increased. These dataindicate that the invasive and inflammatory potential ofS. typhimurium is significantly reduced in the presence ofantimicrobial peptides.

Experiments of coinfection of untransfected cells withS. typhimurium and probiotic E. faecium showed that inthe presence of E. faecium, Salmonella infection caused amuch less intense inflammatory response, and this datais confirmed by invasive assays in which the presence ofE. faecium results in a reduction in the internalization ofS. typhimurium by 45.8%. However, the more interestingresult is that the decrease in the level of inflammatoryresponse due to the presence of E. faecium is furtherreduced in the transfected cells, that is, in the presenceof high concentrations of antimicrobial peptides, suggest-ing that antimicrobial peptides may enhance the beneficialprobiotic activity.

In our experimental system, the ability of AMPs to sig-nificantly reduce the inflammatory response in infectedand coinfected cells is also due to their killing activityagainst Salmonella, as also demonstrated by the count ofCFUs/ml following coinfection, in which the concentrationof pathogen is considerably reduced in the presence ofAMPs with respect to untransfected cells. AMPs could beconsidered, in the future, as a new class of therapeuticssince they are able to induce lesser resistance and have aselective antimicrobial activity to protect the host without

Table 2: CFUs/ml of S. typhimurium and E. faecium in supernatants of coinfected cells.

Inoculum Untransfected hBD-2-transfected hBD-3-transfected

S. typhimurium 1× 107 2× 106 5× 104 4,3× 103

E. faecium 3× 108 3× 108 3× 108 2× 108

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. fae

cium

Figure 4: S. typhimurium internalization assay. UntransfectedCaco-2 cells were infected with S. typhimurium alone orcoinfected with live or heat-killed E. faecium for 4 hours. Thenumber of internalized bacteria was determined by host cell lysis,plating, and counting CFU/well. The data shown arerepresentative of three different experiments± SD. Error barsrepresent standard deviations.


the need for the immune system memory [53]. Having anin vitro system that will produce these proteins will allowus to better clarify the mechanisms underlying these differ-ent behaviors.

Conflicts of Interest

The authors declare that they have no conflict of interestregarding the publication of this article.

Acknowledgments

This study was supported by the MIUR, ProjectPON03PE_00060_3.

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